High levels of homocysteine downregulate apolipoprotein E expression via nuclear factor kappa B

AIM: To investigate the effect of high homocysteine(Hcy) levels on apolipoprotein E(apoE) expression and the signaling pathways involved in this gene regulation.METHODS: Reverse transcriptase polymerase chain reaction(RT-PCR) and Western blot were used to assess apo E expression in cells treated with various concentrations(50-500 μmol/L) of Hcy. Calcium phosphatetransient transfections were performed in HEK-293 and RAW 264.7 cells to evaluate the effect of Hcy on apoE regulatory elements [promoter and distal multienhancer 2(ME2)]. To this aim, plasmids containing the proximal apoE promoter [(-500/+73)apoE construct] alone or in the presence of ME2 [ME2/(-500/+73)apoE construct] to drive the expression of the reporter luciferase gene were used. Co-transfection experiments were carried out to investigate the downstream effectors of Hcymediated regulation of apoE promoter by using specific inhibitors or a dominant negative form of IKβ. In other co-transfections, the luciferase reporter was under the control of synthetic promoters containing multiple specific binding sites for nuclear factor kappa B(NF-κB), activator protein-1(AP-1) or nuclear factor of activated T cells(NFAT). Chromatin immunoprecipitation(ChI P)assay was accomplished to detect the binding of NF-κB p65 subunit to the apoE promoter in HEK-293 treated with 500 μmol/L Hcy. As control, cells were incubated with similar concentration of cysteine. NF-κB p65 proteins bound to DNA were immunoprecipitated with anti-p65 antibodies and DNA was identified by PCR using primers amplifying the region-100/+4 of the apoE gene. RESULTS: RT-PCR revealed that high levels of Hcy(250-750 μmol/L) induced a 2-3 fold decrease in apoE m RNA levels in HEK-293 cells, while apo E gene expression was not significantly affected by treatment with lower concentrations of Hcy(100 μmol/L). Immunoblotting data provided additional evidence for the negative role of Hcy in apoE expression. Hcy decreased apoE promoter activity, in the presence or absence of ME2, in a dose dependent manner, in both RAW 264.7 and HEK-293 cells, as revealed by transient transfection experiments. The downstream effectors of the signaling pathways of Hcy were also investigated. The inhibitory effect of Hcy on the apo E promoter activity was counteracted by MAPK/ERK kinase 1/2(MEK1/2) inhibitor U0126, suggesting that MEK1/2 is involved in the downregulation of apoE promoter activity by Hcy. Our data demonstrated that Hcy-induced inhibition of apoE took place through activation of NF-κB. Moreover, we demonstrated that Hcy activated a synthetic promoter containing three NF-κB binding sites, but did not affect promoters containing AP-1 or NFAT binding sites. ChI P experiments revealed that NF-κB p65 subunit is recruited to the apoE promoter following Hcy treatment of cells.CONCLUSION: Hcy-induced stress negatively modulates apoE expression via MEK1/2 and NF-κB activation. The decreased apo E expression in peripheral tissues may aggravate atherosclerosis, neurodegenerative diseases and renal dysfunctions.

Neurotrophic and metabotrophic potential of nerve growth factor and brain-derived neurotrophic factor: Linking cardiometabolic and neuropsychiatric diseases

One of biggest recent achievements of neurobiology is the study on neurotrophic factors. The neurotrophins are exciting examples of these factors. They belong to a family of proteins consisting of nerve growth factor(NGF), brain-derived neurotrophic factor(BDNF), neurotrophin-3(NT-3), NT-4/5, NT-6, and NT-7. Today, NGF and BDNF are well recognized to mediate a dizzying number of trophobiological effects, ranging from neurotrophic through immunotrophic and epitheliotrophic to metabotrophic effects. These are implicated in the pathogenesis of various diseases. In the same vein, recent studies in adipobiology reveal that this tissue is the body's largest endocrine and paracrine organ producing multiple signaling proteins collectively termed adipokines, with NGF and BDNF being also produced from adipose tissue. Altogether, neurobiology and adipobiology contribute to the improvement of our knowledge on diseases beyond obesity such as cardiometabolic(atherosclerosis, type 2 diabetes, and metabolic syndrome) and neuropsychiatric(e.g., Alzheimer's disease and depression) diseases. The present review updates evidence for(1) neurotrophic and metabotrophic potentials of NGF and BDNF linking the pathogenesis of these diseases, and(2) NGF- and BDNF-mediated effects in ampakines, NMDA receptor antagonists, antidepressants, selective deacetylase inhibitors, statins, peroxisome proliferator-activated receptor gamma agonists, and purinergic P2X3 receptor up-regulation. This may help to construct a novel paradigm in the field of translational pharmacology of neuro-metabotrophins, particularly NGF and BDNF.

Expression of erB4/HER4 in Gastric Carcinoma

在胃的癌探索 HER4 的表示并且阐明的目的在之间的关系它的过去表示和临床病理学。胃的癌的 68 件样品全部的方法 A 被免疫为 HER4 的表示检验组织化学的试金。结果 HER4 完了在 79.41% 胃的癌表示了。过去 HER4 的表示仅仅与淋巴相关节点转移和 TNM 阶段。结论 erbB4 是基因之一调整胃的癌和人工的干扰的生长过去 HER4 的表示可以是胃的癌的有效治疗。

Patient-specific induced pluripotent stem cells as“disease-in-adish”models for inherited cardiomyopathies and channelopathies–15 years of research

Among inherited cardiac conditions,a special place is kept by cardiomyopathies(CMPs)and channelopathies(CNPs),which pose a substantial healthcare burden due to the complexity of the therapeutic management and cause early mortality.Like other inherited cardiac conditions,genetic CMPs and CNPs exhibit incomplete penetrance and variable expressivity even within carriers of the same pathogenic deoxyribonucleic acid variant,challenging our understanding of the underlying pathogenic mechanisms.Until recently,the lack of accurate physiological preclinical models hindered the investigation of fundamental cellular and molecular mechanisms.The advent of induced pluripotent stem cell(iPSC)technology,along with advances in gene editing,offered unprecedented opportunities to explore hereditary CMPs and CNPs.Hallmark features of iPSCs include the ability to differentiate into unlimited numbers of cells from any of the three germ layers,genetic identity with the subject from whom they were derived,and ease of gene editing,all of which were used to generate“disease-in-a-dish”models of monogenic cardiac conditions.Functionally,iPSC-derived cardiomyocytes that faithfully recapitulate the patient-specific phenotype,allowed the study of disease mechanisms in an individual-/allele-specific manner,as well as the customization of therapeutic regimen.This review provides a synopsis of the most important iPSC-based models of CMPs and CNPs and the potential use for modeling disease mechanisms,personalized therapy and deoxyribonucleic acid variant functional annotation.

Knockdown of apoptosis-inducing factor disrupts function of respiratory complex I

Recent findings suggest that apoptotic protein apoptosis-inducing factor (AIF) may also play an important non-apoptotic function inside mitochondria. AIF was proposed to be an important component of respiratory chain complex I that is the major producer of superoxide radical. The possible role of AIF is still controversial. Superoxide production could be used as a valuable measure of complex I function, because the majority of superoxide is produced there. Therefore, we employed superoxide-specific mitochondrial fluo- rescence dye for detection of superoxide production. We studied an impact of AIF knockdown on function of mitochondrial complex I by analyzing superoxide production in selected cell lines. Our results show that tumoral telomerase-positive (TP) AIF knockdown cell lines display significant increase in superoxide pro- duction in comparison to control cells, while a non-tumoral cell line and tumoral telomerase-negative cell lines with alternative lengthening of telomeres (ALT) show a decrease in superoxide production. According to these results, we can conclude that AIF knockdown disrupts function of complex I and therefore increases the superoxide production in mitochondria. The distinct effect of AIF depletion in various cell lines could result from recently discovered activity of telomerase in mitochondria of TP cancer cells, but this hypothesis needs further investigation.

Effect of Unilateral Low-Frequency Stimulation of Hippocampus on Rapid Kindling—Induced Seizure Development in Rats

Since the last decade deep brain stimulation has been proposed as an alternative treatment for patients who do not become seizure-free with the current pharmacological treatments and cannot undergo resective surgical procedure. However, the optimal stimulation parameters remain undetermined and active research in humans and animals is necessary. The present study was designed to investigate the effect of unilateral Low Frequency Stimulation (LFS) of hippocampus on seizure development by using the hippocampal rapid kindling method (hRK) in rats. We used male Wistar rats implanted with electrodes in the ventral hippocampus. All rats underwent hRK (biphasic square wave pulses, 20 Hz for 10 seconds) during three consecutive days (twelve stimulations per day). The control group (hRK;n = 6) received only RK stimulus, while the treated group (LFS-hRK;n = 8) received also LFS (biphasic square wave pulses, 1 Hz for 30 seconds) immediately before the RK stimulus, during three consecutive days. At the end of behavioral testing on day 3, 62% (P < 0.05) of the animals receiving LFS treatment were still not fully kindled staying in stages 0-III (P < 0.01). The number of stimulations needed to achieve generalized seizures (stage IV-V of Racine scale) was significantly higher (P < 0.05) in the LFS group with respect to control group. No significant differences in the cumulative daily afterdischarge duration were observed between both groups. These findings suggest that preemptive LFS can significantly decrease the incidence of hippocampus-kindled seizures and delay the progression and secondary generalization of focal seizures.

Hippocampal High-Frequency Stimulation Inhibites the Progression of Rapid Kindling-Induced Seizure in Rats

Epilepsy is one of the most common serious neurological disorders. Pharmacoresistant epilepsy patients are poorly controlled or their seizures are refractory to drug treatment. Resective surgery is frequently a promising therapy in this population, however, not all the patients meet the eligibility criteria for the surgical treatment. Deep brain stimulation has been investigated in clinical studies and animal studies as an alternative treatment, but the optimal stimulation parameters remain an issue. The present study was designed to investigate the effect of unilateral high-frequency stimulation (HFS) of hippocampus on seizure development by using the hippocampal rapid kindling method (hRK) in rats, and compared the results with those of low-frequency stimulation previously published by our group. We used male Wistar rats implanted with electrodes in the ventral hippocampus. All rats underwent hRK (biphasic square wave pulses, 20 Hz for 10 seconds) during three consecutive days (twelve stimulations per day). The control group (hRK;n = 7) received only RK stimulus, while the treated group (HFS-hRK;n = 9) received also HFS (biphasic square wave pulses, 130 Hz for 30 seconds) immediately before the RK stimulus, during three consecutive days. At the end of behavioral testing 78% (p 0.01) of the animals receiving HFS treatment were still not fully kindled staying in stages 0 -III (p 0.01). HFS group needed a higher number of stimulations to achieve stage III (p 0.05) with respect to control group. However, no significant differences in the cumulative daily afterdischarge duration were observed. HFS did not present significant differences compared with LFS in any of studied parameters. The findings suggest that unilateral HFS applied on hippocampus effectively inhibited the epileptogenic process induced by hippocampal rapid kindling. According to the comparative results about hippocampal rapid kindled animals stimulated with HFS and LFS (5 Hz), we found no conclusive information on which treatment is most efficient.

Vascular dysfunction in diabetes: The endothelial progenitor cells as new therapeutic strategy

The vascular endothelium is a critical determinant of dia- betes-associated vascular complications, and improving endothelial function is an important target for therapy. Diabetes mellitus contributes to endothelial cell injury and dysfunction. Endothelial progenitor cells (EPCs) play a critical role in maintaining endothelial function and might affect the progression of vascular disease. EPCs are essential to blood vessel formation, can differentiate into mature endothelial cells, and promote the repair of damaged endothelium. In diabetes, the circulating EPC count is low and their functionality is impaired. The me- chanisms that underlie this reduced count and impaired functionality are poorly understood. Knowledge of the status of EPCs is critical for assessing the health of the vascular system, and interventions that increase the number of EPCs and restore their angiogenic activity in diabetes may prove to be particularly beneficial. The pre-sent review outlines current thinking on EPCs' therapeutic potential in endothelial dysfunction in diabetes, as well as evidence-based perspectives regarding their use for vascular regenerative medicine.

erbB4/HER4在非小细胞NSCLC中的表达研究(英文)

Objective: To test the expression of HER4 in non-small cell lung cancer (NSCLC) and elucidate the relationship between its over-expression and the clinical pathology of NSCLC. Methods: 70 cases of paraffin-embedded tissues from informative NSCLC were tested for the expression of HER4 by means of immunohistochemical assay. Results: HER4 were overexpressed in NSCLC in 91.4%. The overexpression of HER4 correlated only with the lymph node metastasis, TNM staging and survival after operation. Conclusion: ErbB4 is one of the genes to regulate the growth of NSCLC in advanced stages and artificial interference of the overexpression of HER4 in NSCLC might be a good way for the treatment of NSCLC in advanced stages.

Circulating microparticles and micro RNAs as players in atherosclerosis

Microparticles(MPs) are extracellular membrane vesicles released from normal, apoptotic and pathological cells following a process of detachment from cells of origin. MPs are typically defined by their size, exposure of phosphatidylserine, the expression of surface antigens, proteins and genetic material, originating from their donor cells, and as important vehicles of intercellular communication across numerous biological processes. MPs contain the major source of systemic RNA including micro RNA(mi RNA) of which aberrant expression appears to be associated with stage and progression of atherosclerosis. The involvement and influence of mi RNA during the onset and progression of atherosclerotic disease have generated a lot of inter-est in assessing the feasibility of therapeutic regulation of mi RNAs to manipulate them with a special focus on cardiovascular disease. We speculate on the future de velopments of MPs which contain mi RNA as new thera peutic targets for proliferative vascular diseases such as atherosclerosis.

Surface functionalization of acrylic based photocrosslinkable resin for 3D printing applications

The limited number of resins,available for stereolithography applications,is one of the key drivers in research applied to rapid prototyping.In this work an acrylic photocrosslinkable resin based on methyl methacrylate(MMA),butyl methacrylate(BMA)and poly(ethylene glycol)dimethacrylate(PEGDA)was developed with different composition and characterized in terms of mechanical,thermal and biological behaviour.Two different systems have been developed using different amount of reagent.The influence of every components have been evaluated on the final characteristic of the resin in order to optimize the final composition for applications in bone tissue engineering.The crosslinked materials showed good mechanical properties and thermal stabilities and moreover cytotoxicity test confirms good biocompatibility with no cytotoxic effect on cells metabolism.Moreover two different treatments have been proposed,using fetal bovine serum(FBS)and methanol(MeOH),in order to improve cell recognition of the surfaces.Samples threatened with MeOH allow cell adhesion and survival,promoting spreading,elongation and fusion of C2C12 muscle myoblast cells.

Hepatic Alarmins and Mitochondrial Dysfunction under Residual Hyperlipidemic Stress Lead to Irreversible NAFLD

Background and Aims:Nonalcoholic fatty liver disease(NAFLD)includes a range of progressive disorders generated by excess lipid accumulation in the liver leading to hepatic steatosis and eventually fibrosis.We aimed to identify by high performance mass spectrometry-based proteomics the main signaling pathways and liver proteome changes induced by hypercholesterolemia in a rabbit atherosclerotic model that induced high accumulation of lipids in the liver.Methods:The effect of combined lipid-lowering drugs(statins and anti-PCSK9 monoclonal antibody)were used after the interruption of the hypercholesterolemic diet to identify also the potential mediators,such as alarmins,responsible for the irreversible NAFLD build up under the hyperlipidemic sustained stress.Results:Proteomic analysis revealed a number of proteins whose abundance was altered.They were components of metabolic pathways including fatty-acid degradation,glycolysis/gluconeogenesis,and nonalcoholic fatty liver disease.Mitochondrial dysfunction indicated alteration at the mitochondrial respiratory chain level and down-regulation of NADH:ubiquinone oxidoreductase.The expression of a majority of cytochromes(P4502E1,b5,and c)were up-regulated by lipid-lowering treatment.Long-term hyperlipidemic stress,even with a low-fat diet and lipid-lowering treatment,was accompanied by alarmin release(annexins,galectins,HSPs,HMGB1,S100 proteins,calreticulin,and fibronectin)that generated local inflammation and induced liver steatosis and aggressive fibrosis(by high abundance of galectin 3,fibronectin,and calreticulin).Conclusions:The novel findings of this study were related to the residual effects of hyperlipidemic stress with consistent,combined lipid-lowering treatment with statin and inhibitor of PCSK9.